Selection and/or enhancement of resident microorganisms in the gastrointestinal tract

ABSTRACT

Improved method of enhancing a population of one or more target microorganisms in the gastrointestinal tract of an animal, the improvement comprising providing to the animal a selected modified or unmodified resistant starch or mixtures thereof, such that the one or more microorganisms will selectively utilise the starch and/or increase in number and/or activity in the gastrointestinal tract, either uniformly throughout the gastrointestinal tract or at specific site or regions.

TECHNICAL FIELD

This invention relates to an improved method of enhancing a populationof one or more target microorganisms in the gastrointestinal tract,especially the small intestine and the large bowel, of animals andhumans.

BACKGROUND ART

It is the contention of many scientists that the health and well beingof people can be positively or negatively influenced by themicroorganisms which inhabit the gastrointestinal tract, and inparticular, the large bowel. These microorganisms through the productionof toxins, metabolic by-products, short chain fatty acids, and the likeaffect the physiological condition of the host. The constitution andquantity of the gut microflora can be influenced by conditions or stressinduced by disease, life style, travel, and other factors. Ifmicroorganisms which positively affect the health and well being of theindividual can be encouraged to populate the large bowel, this shouldimprove the physiological well being of the host

The present inventors have realised that it would be desirable toprovide a medium that would function to promote the growth and/oractivity of target microorganisms in the gastrointestinal tract ofanimals including humans.

DISCLOSURE OF INVENTION

The present invention consists in an improved method of enhancing apopulation of one or more target microorganisms in the gastrointestinaltract of an animal. the improvement comprising providing to the animal aselected modified or unmodified resistant starch or mixtures thereof,such that the one or more microorganisms will selectively utilise thestarch and/or increase in number and/or activity in the gastrointestinaltract.

The target population of microorganism may be enhanced throughout thegastrointestinal tract of the animal or targeted at specific sites ofthe gastrointestinal tract. It will be appreciated that the presentinvention will be suitable for any animal that requires alteration ofits gastrointestinal flora. The present method is particularly suitablefor use in humans.

The starches suitable include resistant or high amylose starches andmodified forms thereof. The animal or human may be fed the selectedresistant starch or the starch may be incorporated in a probioticcomposition.

As used in this specification, “resistant starch” includes those formsdefined as RS1, RS2, RS3 and RS4 as defined in Brown, McNaught andMoloney (1995) Food Australia 47: 272-275. Either modified or unmodifiedresistant starches or mixtures thereof are used in this invention. Theadvantage of resistant starch is that it is largely not degraded untilit reaches the large bowel. Therefore it provides a readily availablesubstrate for fermentation by the target microorganisms as soon as theyarrive in the large bowel. In both cases, a preferred form of resistantstarch is a high amylose starch particularly high amylose starches asdisclosed and taught in WO 94/03049 and WO 94/14342, the contents ofwhich are incorporated into this specification for the purposes ofconvenient cross-reference.

In WO 94103049 and WO 94/14342, high amylose starches are disclosedwhich are resistant starches and include maize starch having an amylosecontent of 50% w/w or more, particularly 80% w/w or more, rice or wheatstarch having an amylose content of 27% w/w or more and; particulargranular size ranges of starches having an amylose content of 50% ormore and enhanced resistant starch content, these starches includingmaize, barley, and legumes. This invention is not, however, limited tothese forms of resistant starch. For example, other forms of resistantstarch are derived from sources such as bananas and tubers such aspotatoes and modified forms thereof.

It may be advantageous to also chemically modify the starch to, forinstance, alter the charge density or hydrophobicity of the granuleand/or granule surface to enhance the attachment compatibility betweenthe microorganism and the resistant starch. Chemical modifications, suchas etherification, esterification, acidification and the like are wellknown in this art as being suitable chemical treatments.

To modify the degree of enzyme susceptibility of the resistant starchthe conformation or structure of the starch can be altered. Examplesinclude acid or enzyme thinning and cross bonding using difunctionalreagents.

The starches may be modified physically by, for example,crystallisation.

It is also within the scope of this invention to subject enzymaticallytreated resistant starches to chemical modification as described above.

As used herein, Hi-maize™ (trade mark) refers to a high amylose starchobtained from Starch Australasia Limited.

In order that the present invention may be more clearly understood,preferred forms thereof will be described with reference to thefollowing figure and examples.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows comparison of the co-culturing of Lactobacillus acidophiluswith Bifidobacterium strain X8AT2 in glucose and amylose starch medium.

FIG. 2 shows enumeration of number of bifidobacteria in starch basedmedium inoculated with human faecal homogenates and incubatedanaerobically at 37° C. for 12 hours. Individual starches according tothe description in Table 4.

FIG. 3 shows enumeration of number of amylolytic bacteria in starchbased media inoculated with human faecal homogenates and incubatedanaerobically at 37° C. for 12 hours. Individual starches as in Table 4.

FIG. 4 shows enumeration of major bacterial groups in stomach contentsfrom mice on various starch based diets (Table 4).

FIG. 5 shows enumeration of major bacterial groups in ileal contentsfrom mice on various starch based diets (Table 4).

FIG. 6 shows enumeration of major bacterial groups caecal contents frommice on various starch based diets (Table 4)

FIG. 7 shows enumeration of major bacterial group in colon contents frommice on various starch based diets (Table 4).

FIG. 8 shows the total anaerobic microbial population of ileal origin, 9hours post inoculation in media containing starch nos 4, 6, 8, 9 andglucose.

FIG. 9 shows the total anaerobic microbial population of caecal origin,9 hours post inoculation in media containing starch nos 4, 6, 8, 9 andglucose.

MODES FOR CARRYING OUT THE INVENTION EXAMPLE 1

By measuring the amylase activity of specific intestinal bacteria whengrown in standard laboratory medium containing glucose, starch(amylopectin) or resistant starch (amylose) added to a defined medium(composition included in Table 1 at a final concentration of 10 mg/ml),one can show that many of the intestinal bacteria produce amylase whichcan utilise the resistant starch (Table 2). In addition, the specificgrowth rates when six different intestinal bacteria were grown onglucose, amylose, amylopectin, Hi-maize™ and carboxymethylated resistantstarch were determined (Table 3). The various bacteria tested grew atvery different rates to each other, indicative that individual bacterialgroups or species will be selectively enhanced by the form of starchused.

TABLE 1 Composition of medium used for growing intestinal strains ofbacteria. Ingredient Amount Bacteriological peptone 7.5 g Yeast extract2.5 g Tryptone 5.0 g Starch 10.0 g K₂HPO₄ 2.0 g KH₂PO₄ 1.0 g NaHCO₃ 0.2g NaCl₂ 2.0 g MgCl₂ 0.2 CaCl₂ 0.2 g MnCl₂ 0.02 g CoCl₂ 0.02 g Cystein0.5 g FeSO₄ 0.005 g Tween 80 2 ml Hemin 0.005 g Vit B₁₂ 0.001 g Vit K0.0005 g Water (final volume) 1 liter

TABLE 2 Amylase activity after growth of intestinal isolates on starchand resistant starch. Bacteria Glucose Amylopectin Amylose 1.Supernatant Cl. butyricum 0.592 0.987 0.317 Bact. fragilis 0.064 0.5630.927 Bif. bifidum 0.506 0.131 0.293 Bif. pseudolongum 0.087 0.542 0.423E. limosum 0.202 0.568 0.794 Bact. vulgatus 0.196 0.602 0.380 2. CellExtract Cl. butyricum 0.000 0.000 0.021 Bact. fragilis 0.045 1.038 2.018Bif. bifidum 0.295 4.271 9.270 Bif. pseudolongum 0.664 3.855 12.685 E.limosum 0.375 0.491 0.039 Bact. vulgatus 0.229 1.644 3.381

TABLE 2 Amylase activity after growth of intestinal isolates on starchand resistant starch. Bacteria Glucose Amylopectin Amylose 1.Supernatant Cl. butyricum 0.592 0.987 0.317 Bact. fragilis 0.064 0.5630.927 Bif. bifidum 0.506 0.131 0.293 Bif. pseudolongum 0.087 0.542 0.423E. limosum 0.202 0.568 0.794 Bact. vulgatus 0.196 0.602 0.380 2. CellExtract Cl. butyricum 0.000 0.000 0.021 Bact. fragilis 0.045 1.038 2.018Bif. bifidum 0.295 4.271 9.270 Bif. pseudolongum 0.664 3.855 12.685 E.limosum 0.375 0.491 0.039 Bact. vulgatus 0.229 1.644 3.381

EXAMPLE 2

A number of modifications of the resistant starch (Hi-maize™) (Table 4)were used in the defined growth medium presented in Table 1. Theintestinal isolates were then inoculated and the starch concentrationdetermined ned after 22 h incubation as an indication of the extent ofutilisation. Total carbohydrate was estimated using phenol-sulphuricacid assay. Surprisingly, a modification often resulted in alteredutilisation of the starch as can be seen in Table 5.

TABLE 4 Starch identification Starch Destination Identification Analysis1 A939 (D19) Hydroxypropylated DS* = 0.13 2 A938 (C79) Acetylated Acetylvalue = 2.69% 3 A961 (D8) Octenyl succinated OSA value = 4.73% 4 A955(D2) Carboxymethylated Carboxyl value = 1.0% 5 A960 (D7) SuccinatedSuccinyl value = 3.97% 6 HA 008 Unmodified — (D2118) 7 A993 D42Succinated Succinyl value = 4.1% 8 A956 (D1) Carboxymethylated Carboxylvalue = 2.0% 9 A995 (D57) Acetylated Acetyl value = 4.0% 10 A965 (D9)Hydroxypropylated DS = 0.13 *degree of substitution

TABLE 5 Concentration of starch after incubation for 22 hours. StarchesBacteria 1 2 3 4 5 6 7 8 9 10 Cl. butyricum 3.364 1.829 2.354 3.7141.418 2.175 2.980 3.121 2.648 — Bif. pseudolongum 5.532 4.029 5.0913.658 6.843 5.308 5.130 4.157 4.899 4.463 Bif. bifidum 5.245 4.132 7.0354.503 5.437 4.950 4.375 4.720 5.091 5.454 Bact. fragilis 4.081 5.3727.995 4.669 7.547 6.971 6.140 5.001 7.547 5.660 Bact. vulgatus — 8.5707.419 6.843 8.954 9.210 10.489 6.108 6.332 6.908 Bif. strain X8AT210.106 6.492 10.00 5.532 6.268 7.931 9.850 6.843 5.820 5.916 Lact.acidophilus 8.75 10.501 10.50 10.50 92.84 10.50 10.07 10.50 10.50 95.54Lact. helviticus 52.76 10.50 10.50 10.50 10.50 10.50 10.50 99.68 10.5010.50 Starch concentrations after 22 hours incubation (mg/ml) 1: A.939(D19) Hydroxypropylated; 2: A.938 (C79) Acetylated; 3: A.961 (D8)Octenyl succinated; 4: A.955 (D2) Carboxylated; 5: A.960 (D7)Succinated; 6: HA 008 (D2118) Unmodified; 7: A993 D42 Succinated; 8:A956 (D1) Carboxymethylated; 9: A995 (D57) Acetylated; 10: A965 (D9)Hydroxyproplated;

EXAMPLE 3

The effect of Co-culture with Amylolytic Bif. X8AT2 on the Growth ofLactobacillus sp in the Amylose Starch Medium

The growth of Lact. acidophilus in the Hi-maize™ containing medium withor without the present of Bif. X8AT2 was compared. The growth mediumcontained 1% Hi-maize™ starch or glucose as the growth carbon and energysource. The medium was autoclaved at 121° C. for 15 minutes and strictlyanaerobic conditions were used during the medium preparation. Overnightcultures (0.1 ml) of Lact. acidophilus and Bif. X8AT2 were inoculatedinto the serum tubes containing either glucose or Hi-maize™ starch basedmedia. For the control Lact. acidophilus only was inoculated into theserum tubes. The tubes were then incubated at 37° C. for 24 hours.Samples were taken at 0, 2, 4, 6, 8, 10, 12 and 24 hours to enumeratethe population of Lact. acidophilus by using standard series dilutionmethod. The population of Lact. acidophilus was expressed as CFU/ml onMRS agar plates.

Since Lact. acidophilus can not degrade Hi-maize™ starch, the growth ofLact. acidophilus in the defined medium containing Hi-maize™ starch asthe sole carbon source was very slow and resulted in low biomass. Theimprovement of the growth of Lact. acidopliilus in Hi-maize™ medium wasobserved when the strain was co cultured with the Hi-maize™starch-utiliser, Bif. strain X8AT2 (FIG. 1). As can be seen in FIG. 1, asynergistic effect is demonstrated when the Bifidobacterial strain isinoculated with the Lactobacillus.

EXAMPLE 4

Mice were fed either normal mouse diet or a prepared diet containingeither waxy starch, Hi-maize™ or modified Hi-maize™ (carbo.xvmethylated)and were orally dosed with 200 microlitres of Bifidobacterium sp strainX8AT2 or Bifidobacterium bifidum cultures. The composition of the mouseprepared diet is included in Table 6. Faecal samples were collectedafter continuous feeding from day 3 to day 8 of the diet plus thebifidobacteria. The major bacterial roups were enumerated usingselective media and the total bacteria output for the groups werecalculated. As can be seen in Table 7, Bacteroides numbers were enhancedsignificantly in mice when they were fed a modified resistant starchplus bifidobacteria compared to controls, which include mice fedresistant starch plus bifidobacteria. While it is established thatBacteroides of intestinal origin can ferment both starch (amylopectin)and resistant starch (amylose) reviewed by Salyers and Leedle (Salyers &Leedle, 1983), it is surprising to discover that a carboxymethylatedamylose can significantly increase growth of the Bacteroides.

TABLE 6 Diets for mice probiotic feeding experiments. Test Groups A B CD E Starch Waxy HA Carboxy- HA None 400 400 methyl 400 400 Casein 200200 200 200 Canola oil 25 25 25 25 Sunflower oil 25 25 25 25 Sucrose 150150 150 150 Wheat bran 100 100 100 100 Gelatin 20 20 20 20 Mineral mix67 67 67 67 Vitamin mix 13 13 13 13 Methionine 2 2 2 2 Bacterial strainX8AT2 X8AT2 X8AT2 None X8AT2 Waxy = waxy maize; HA = High amylosestarch; Carboxy-methyl = Carboxymethylated high amylose starch. Allweights are in grams. Bacterial cultures (100 microliters per day) wereorally ingested by the mice with starch containing meals.

TABLE 7 Bacteroides population in mice feeding study (total bacteriaoutput per day per mice). Starches Group A Group B Group C Group D GroupE Bacteroides 9.163 ± 8.961 ± 9.952 ± 8.961 ± 8.463 ± Mean Diff. 0.420.40 0.357 0.576 0.569 (compare A-B: A-C: A-D: A-E: to group A) 0.202−0.789 0.202 0.699 F-test none p < 0.05 none p < 0.05 Mean Diff. A-E:E-B: E-C: E-D: (compare −0.699 −0.498 −1.489 −0.497 to group E) F-test p< 0.05 p < 0.05 p < 0.05 p < 0.05 Group A: Waxy starch plus X8AT2 —Bifidobacteria human isolates; Group B: Hi-maize ™ starch plus X8AT2;Group C: Carboxymethylated resistant starch plus X8AT2; Group D:Hi-maize ™ starch plus Bif. bifidum; Group E: Normal mice diet plusX8AT2

EXAMPLE 5

a) Four groups of six mice (Balb/c, SPF) were continuously fed withsemisynthetic diets for 4 weeks. Group A received 40% waxy starch in thediet, and groups C and E had 40% modified starches D2 and D57,respectively in their diets. Group D was the Hi-maize™ starch group andgroup B was assigned as the control to be fed with normal mice diet. Twofaecal samples were collected at the end of experimental period (4weeks) to enumerate the population of Bifidobacterium by using propionicacid agar. Bifidobacteria were further identified by cell morphologyunder light microscopy. The population of Bifidobacterium was expressedas total output per day per mice.

The results from three experiments indicated that the specific pathogenfree (SPF) mice used in the experiment were free of detectablebifidobacteria (<10~3) and continued to be so for the 2 months ascontrol animals (Table 8). It is very surprising, however, to find thatwhen the mice shifted from normal mice diet to the starch diets, thepopulation of bifidobacteria increased significantly. The degree ofincrease depended on the type of starch incorporated into the diets.Hi-maize™ starch diet yielded the greatest numbers of nativebifidobacteria in the mice faeces, followed by the waxy starch dietModified Hi-maize™ starch D57 demonstrated better results in thestimulation of the growth of bifidobacteria than modified Hi-maize™starch D2. The results from previous experiments indicated that D2starch mainly sustained the good growth of Bacteroides. The statisticalanalysis of the data is also presented in Table 8.

After the first stage of experiment in which the mice were fed with theexperimental diet for 4 weeks, 200 ul of Bif. X8AT2 was orally dosedinto mice for 5 days. Numbers of Lactobacillus from all of groups werequantified in the mice faeces at both stages of experiments by usingRogosa agar. The cell morphology of Lactobacillus were also checkedunder phase-contract microscopy.

It can be seen that the highest fermentability of starch was detectedwith Cl. butyrium. Bif. bifidum and Bif. psuedologum are also capable ofhvdrolysilg all of the starches. while human isolate Bif. X8AT2preferred starch nos. 2, 4, 5, 8, 9 and 10. Bact. fragilis has astronger amvlolytic capability to degrade starches than Bact. vulgatus.The poorest genus is Lactobacillus, since both strains tested could onlypartially utilise the modified Hi-maize™ starch 1.

All of the mice were heavily colonised with dense populations ofLactobacillus. The influence of diets on faecal population ofLactobacillus is shown in Table 9. In general, none of the starch dietssupported the increased growth of native Lactobacillus, in comparisonwith normal mice diets. Particularly low numbers of Lactobacillus weredetected in the groups of mice fed with modified starches D2 and D57.The population of Lactobacillus, however, increased in the group of micefed with Hi-maize™ diet when amylolytic bifidobacterial strain XBAT2 wasassociated with the mice.

TABLE 8 Native population of Bifidobacteria in mice fed with differentstarches diets (CFU log 10/g faeces) Starches Group A Group B Group CGroup D Group E Bifido- 7.48 ± 0 ± 0 1.475 ± 8.235 ± 6.432 ± bacterium0.481 2.174 0.46 0.566 Positive 6/6 0/6 2/6 6/6 6/6 mice in the testgroup Mean Diff. A-B: A-C: A-D: A-E: (compare 7.47 −6.005 −0.755 −1.048to group A) F-test p < 0.05 p < 0.05 none none Mean Diff. B-A: B-C: B-D:B-E: (compare −7.48 −1.475 −8.235 −6.432 to group B) F-test p < 0.05 p <0.05 p < 0.05 p < 0.05 Group A: Waxy starch Group B: Normal mice dietGroup C: Carboxymethylated amylose starch Group D: Hi-maize ™ starchGroup E: Acetylated maize starch

TABLE 9 Lactobacillus population in the mice fed with different starchesdiets (CFU log 10/g wet faeces) Starches Group A Group B Group C Group DGroup E Lactobacillus Period 1: Fed with 7.596 ± 8.113 ± 7.423 ± 7.858 ±7.309 ± experimental 0.477 0.532 0.295 0.367 0.326 diets for 4 weeksMean Diff. B-A: B-C: B-D: B-E: (compare 0.517 −0.690 −0.255 −0.804 togroup B) F-test none p < 0.05 none p < 0.05 Period 2: Experimental 7.823± 7.782 ± 7.501 ± 8.031 ± 7.451 ± diets plus 0.397 0.477 0.319 0.5290.673 Bifido- bacterium X8AT2 Mean Diff. D-A: D-B: D-C: D-E: (compare0.208 0.249 0.531 −0.580 to group D) F-test none none p < 0.05 p < 0.05

EXAMPLE 6

a) Material from human colon was diluted Wilkins Chargren broth(1:1000).

The mixtures were incubated 37° C. for 24 h and sampled at 0, 3, 6, 9,12 and 24 h post inoculation.

The type of resident starch or modifications thereof will induce analteration or stimulation of resident microbes. After 9 h incubation,Starch nos. 8 and 9. induced an increase in the bifidobacterialpopulation (FIG. 2) followed by the bifidobacterial populations ofcultures supplemented with Starch no. 1, 2, 10 and 7. Culturessupplemented with Starch no. 6 were less benefited, resulting in arelatively poor development of the bifidobacterial population. Starchno. 3 had only a moderate beneficial effect on bifidobacterial growth.

A large stimulation of the amylolytic microbial population (FIG. 3) wasdetected when either Starch nos. 8, 4, 10 or 9 were used as a source ofcarbon. In contrast, poor development of the bifidobacterial populationwas noted in cultures supplied with Starch nos. 6, 3, 7 and 5. A closecorrelation between growth response of amylolytic and bifidobacterialpopulations was noted (FIGS. 2 and 3).

EXAMPLE 7

Degradation of Starch nos. 1-10 by Human Faecal Microorganisms

The degradation of resistant starch and modifications thereof (Table 4)by human faecal microbes was studied. After 12 and 24 h incubation offaecal homogenates in media based on the starches in Table 4 the variousdegree of utilisation was determined (Table 10). There was a greatvariation in resistance to degradation. Starch nos. 1 and 8 were mostefficiently degraded by the human faecal microbiota, which resulted in0.31 and 1.8%, respectively starch remaining in the cultures 24 h postinoculation. Starch nos. 7 and 9 were less efficiently degraded, givingabout 9% remaining starch in the final culture 24 h post inoculation.The most resistant starch was Starch no. 6. The difference in resistanceto degradation was even more significant in cultures incubated for 12 h.At this point (12 h), six starches were assayed: Starch nos. 1, 4, 6, 7,8 and 9. Starch no. 1 was of the starches the most easily degraded(2.74% remaining), followed by Starch no. 8 (5.3%). Starch no. 4(23.4%). Starch no. 9 (44.50%), Starch no. 7 (79.7%) and Starch no. 6,the one most resistant to degradation. No degradation of starch no. 6could be detected 12 h post inoculation (Table 10).

TABLE 10 Degradation of Starch nos. 1-10 by human faecal microorganisms.Type of Starch Residual starch (%) (Table 4) 12 h post inoculation 24 hpost inoculation 1 2.73 ± 0.46 0.31 ± 0.10 2 N/A 7.07 ± 1.24 3 N/A 8.57± 1.08 4 23.4 ± 4.72 5.59 ± 1.73 5 N/A 11.8 ± 2.86 6 119. ± 17.4 29.9 ±8.57 7 79.2 ± 11.3 9.01 ± 2.85 8 5.26 ± 1.48 1.76 ± 0.34 9 44.5 ± 1.587.55 ± 0.95 10 N/A 9.38 ± 1.80

EXAMPLE 8

Hi-maize™ can be modified to various levels with chemical reagents, suchas acetic anhydride. The degree of susceptibility to in vitro digestionby bacterial alpha-amylase and amyloglucosidase of Hi-maize™ and threeacetylated starches from Hi-Maize™ was ascertained using the MegazymeTotal Starch Assay Procedure (AA/AMG 6/95). Each starch was solubilisedand the enzyme resistant “residue” recovered by centrifugation. Theresidue was then solubilised using DMSO and assayed as per the Megazymeresistant starch method. The results are shown in Table 11.

TABLE 11 Resistance of acetylated Hi-maize ™ starch to amylase digestionEnzyme Amylose Acetyl solubilised Starch content value starch residueStarch type (%) dsb* (%) dsb (%) dsb (%) dsb Hi-maize ™ 85 0 93.8 6.2Starch A — 2.85 66.5 33.5 Starch B — 4.39 58.5 41.5 Starch C — 7.72 35.564.5 *dry solids basis

TABLE 11 Resistance of acetylated Hi-maize ™ starch to amylase digestionEnzyme Amylose Acetyl solubilised Starch content value starch residueStarch type (%) dsb* (%) dsb (%) dsb (%) dsb Hi-maize ™ 85 0 93.8 6.2Starch A — 2.85 66.5 33.5 Starch B — 4.39 58.5 41.5 Starch C — 7.72 35.564.5 *dry solids basis

EXAMPLE 9

This example demonstrates that various modifications of resistant starchas presented in Table 4 induce the development of microbes with varyingamylolytic activity. (Starch no. 1 and 8 are soluble and could not beassessed in this study). This was assessed by relating the number ofisolates that produced clearing zones on amylose agar to the totalpopulation (CFU) on amylose plates (in % of total), and the degree ofamylolytic activity expressed by amylolytic isolates. This was assessedby measuring clearing zones developed around colonies with amylolyticactivity. There was a great variation in capacity the human faecalmicrobiota to degrade the different starches. Starch nos. 2 and 3 weredegraded by the highest percentage of the population (65%) followed byStarch no 8 which was degraded by 56% of the population (Table 12).Starches 3 and 6 were degraded by only 12% and 7% respectively.

Production of Short Chain Fatly Acids (SCFA)

Compared to the glucose control, the addition of starches (except Starchno. 11) resulted in a significant increase in the production of allinvestigated SCFA's.

The production of n-butyric acid was greatest in media containing Starchno. 8, followed by Starch no. 4, Starch no. 5, Starch no. 2, Starch no.6 and media containing Starch no. 10 (Table 13).

The production of acetic acid was greatest in media containing Starchno. 8, followed by Starch no. 1, Starch no. 2, Starch no. 10, Starch no.5 and media containing Starch no. 9.

The production of propionic acid was greatest in media containing Starchno. 8, followed by Starch no. 3, Starch no. 9, Starch no. 6, Starch no.4 and media containing Starch no. 2.

The production of iso-butyric acid was greatest in media containingglucose, followed by Starch no. 7 and Starch no. 3. Iso-butyric acidcould not be detected in cultures supplied with any other starches.

The production of iso-valeric acid was greatest in media containingStarch no. 6, followed by media containing Starch no. 4, Starch no. 9,Starch no. 8, Starch no. 5 and glucose.

Starch no. 8 promoted production of all major SCFA's (acetic, propionicand butyric acid), more that any of the other starch, that resulted in abutyric acid concentration that was about 1.5 times greater than forStarch no. 3 (Table 13).

TABLE 13 Production of Short Chain Fatty Acids from Starch nos. 1-11 andglucose, 24 h post inoculation with human faecal material. Short ChainFatty Acid (mM) Type of carbon iso- iso- source Acetic Propionic Butyricn-Butyric Valeric Starch 1 40.7 ± 15.4 ± 0 11.5 ± 0.18 ± 4.17 1.21 2.440.37 Starch 2 37.9 ± 15.8 ± 0 9.66 ± 0 0.44 0.11 0.37 Starch 3 35.4 ±18.5 ± 0.48 ± 8.28 ± 0.35 ± 0.95 0.62 0.95 0.51 0.40 Starch 4 34.8 ±16.2 ± 0 10.7 ± 0.53 ± 0.71 0.36 0.34 0.46 Starch 5 37.0 ± 15.7 ± 0 10.4± 0.46 ± 7.85 4.65 3.19 0.40 Starch 6 35.8 17.4 0 9.77 1.05 Starch 734.1 ± 15.2 ± 0.79 ± 8.44 ± 0.30 ± 3.35 0.36 1.11 0.07 0.42 Starch 854.4 ± 19.0 ± 0 12.7 ± 0.48 ± 1.65 0.33 1.01 0.68 Starch 9 36.4 ± 17.7 ±0 8.41 ± 0.97 ± 0.90 0.43 0.17 0.02 Starch 10 37.6 ± 15.5 ± 0 9.51 ± 00.82 0.82 0.50 Starch 11 22.5 ± 10.0 ± 0 4.74 ± 0 0.26 0.77 0.25 Glucose31.0 ± 12.4 ± 3.05 ± 7.15 ± 0.41 ± 0.08 1.09 0.30 0.92 0.57

TABLE 14 Efficiency of starch degradation by the microbiota thatcolonises animals fed either Waxy starch, Starch nos. 4, 6 or 9. Animalsfed Degradation of dietary starch Amylose Starch: Starch 4 Starch 6Starch 9 (Sigma) 1 + + +++ + 1 + − + − 4 +++ ++ ++ + 4 ++++ +++ ++ ++ 6++++ ++++ ++++ ++ 6 ++++ + +++ +++ 9 +++ + ++++ +++

EXAMPLE 10

Specific pathogen free (SPF) mice were fed synthetic diets consistentwith those presented in Table 6 but using waxy starch and starches 4, 6and 9 (Table 4). Five animals per group were used and maintained in thediet for 2 weeks. Animals were sacrificed and the gastrointestinal tractwas collected. Contents from the stomach, ileum, caecum and colon werecollected, weighed and stored on ice for processing within an hour. Themajor bacterial groups were enumerated using routine selective media.The groups include the obligate anaerobes, lactobacilli, enterococci,coliforms. amylolytic bacteria, clostridia and bifidobacteria.Amylolytic activity was assessed for isolates from the mice on thevarious diets by ranking the zone of clearance around colonies on agarplates prepared using either amylose (Sigma) of starches 4, 6 or 9.Results are presented in FIGS. 4, 5, 6 and 7. It can be seen in thesefigures that the different starches will induce altered levels ofspecific groups of microbes at different sites in the tract. For examplestarch 6 and 4 stimulate lactobacillus from the stomach, ileum andcaecum; starch 9 stimulates bifidobacterium in all sites samples; starch4 stimulates endospore forming populations such as the clostridia in allsites sampled and suppresses the bifidobacterial numbers as all sitessampled; starch 9 suppressed endospore forming populations in allregions sampled.

EXAMPLE 11

Ex-germ free mice colonised with human faecal homogenates were fed acommercial animal diet. Material from gastrointestinal of germ free micecolonised with human microbes (gastric, ileal, caecal and coloniccontent) was diluted in Wilkins Charlgren broth (1/1000). The faecalmicrobial composition of the aninal that served as a source for inoculumis presented in Table 15. Diluted material was used as the inoculum forthe starch media (Table 1) continuing the different resistant starchesin Table 4. The mice gastrointestinal content mixes were sampled at 0and 9 h post inoculation.

The use of different modifications of resistant starches or unmodifiedstarches could be used to control specific populations at differentsites. This has been shown when gut contents from the stomach, ileum,caecum or colon of ex-germ-free mice colonised with human colonmicroflora were collected and inoculated into media containing thevarious starches as in Table 4. The mixtures were incubatedanaerobically at 37° C. The concentrations of the major bacteria groupswere enumerated and these included the total anaerobes, lactobacilli,bifidobacteria. It was shown that the modification influenced the levelsof the different microbes. For example, starch 9 induced higher levelsof obligate anaerobes in the ileum than were induced by starch 8 (FIG.8) while starch 8 promoted higher levels of these obligate anaerobes inthe caecum than were induced by starch 9 (FIG. 9).

TABLE 15 Microbial composition of faeces from mouse to be used BacteriaCFU per g Lactobacilli <10³ Bifidobacteria 1.7 × 10⁵ Enterococci 3.7 ×10⁷ E. coli <10³ Total anaerobes 9.6 × 10⁹ Total amylolytic <10³endospores 3.3 × 10³

The resident bifidobacterial and amylolytic population may be replacedwith new bfidobacterial and amylolytic populations. This will happen ifthe unrodfified Hi-maize™ (Starch no. 6) is supplied. Althoughbifidobacterial and amylolytic populations will be disadvantaged in theshort term, animals fed Starch 6 (fro about 2.5 months) have a densebifidobacterial and amylolytic population through out thegastrointestinal tract (FIGS. 4, 5, 6 and 7 and Table 14).

Uses

It has been shown that carboxymethylated resistant starch consumptionresulted in greater numbers of faecal Bacteroides than unmodifiedresistant starch. It is well established that Bacteroides spp contributeto saccharide degradation in the large intestine, in particularpolysaccharides degradation (Salyers, 1979). This would result in anincrease in short chain fatty acids, which are used as metabolic fuelfor the epithelial mucosa and for the host. In addition, there is aclear link between the levels of butyrate and the incidence of polypsand cancer (Young, 1996). Consequenitly. enhancing bacteroides numberswill lead to increased fermentation which will contribute to intestinalhealth and protect from the risks of colon cancer.

Other chemically modified starches may lead to enhancement of otherbeneficial bacteria in the large intestine. Consequently, one can use amodified resistant starch in the diet to achieve one or all of thefollowing conditions:

i) as a general gut microflora stabiliser:

ii) in clinical conditions related to disturbances e.g. flora relatedirritable bowel syndrome and inflammatory bowel disease, Crohn^(t)sdisease, diarrhoea;

iii) improved intestinal health e.g. of the epithelial mucosa;

iv) immunostimulating activities; and

v) colon cancer

In addition, as discussed by Coates (Coates, 1988), resistant starchingestion can cause a lowering of the pH which will lead to suppressionof bacterial transformation of cholesterol and bile acids, thusaffecting excretion of cholesterol and bile acids. Since the presentinventors have found that modification of the resistant starch affectedutilisation by specific microbes and the bacterial groups that wereenhanced, modifications of the resistant starch could influencecholesterol and bile acid excretion levels.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

What is claimed is:
 1. A method of increasing the number or activity ofa population of one or more microorganisms resident in thegastrointestinal tract of an animal, the method comprising administeringorally to the animal a selected modified or unmodified resistant starchor mixtures thereof, the resistant starch being obtained from a starchhaving an amylose content of 50% w/w or more, or 27% w/w or more if awheat or rice starch, in an amount effective such that the population ofone or more microorganisms utilizes the resistant starch in a mannersuch that the number or activity of the population of one or moremicroorganisms increases in the gastrointestinal tract, wherein themethod further comprises assaying for the levels of the microorganismspresent in the gastrointestinal tract.